RESUMEN

BACKGROUND: Antimicrobial resistance is a major global public health issue. Infections caused by resistant species are associated with higher mortality rates, longer hospital stays, medication failure, and rising medical costs. The World Health Organisation has declared multidrug resistance-associated infections as an epidemic of public health concern. OBJECTIVE: This study aimed to evaluate the antimicrobial resistance profile and associated factors of hospital-acquired Gram-negative bacterial pathogens among hospitalized patients in Northeast Ethiopia. MATERIALS AND METHODS: A health facility-based cross-sectional study was conducted among hospitalized patients from March 2021 to February 2022. About 810 clinical specimens were collected, transported, and processed from admitted patients following the standard bacteriological procedures. The clinical samples were inoculated onto blood agar, MacConkey agar, and chocolate agar. Furthermore, the species identification was done using gram reactions, colony morphology, and color and biochemical tests. Antimicrobial susceptibility tests, extended-spectrum beta-lactamase, and carbapenemase production were performed as per the clinical laboratory standard institute guidelines. For analysis, the information was entered into Epi-data and exported to SPSS. A P value of < 0.05 with a 95% confidence interval was considered as a statistically significant association. RESULTS: Out of 810 clinical specimens, 285/810 (35.2%) developed bacterial infections. From the isolated bacteria, E. coli was the predominant bacteria accounting for 78/285 (27.4%) followed by K. pneumoniae, 69/285(24.42%), whereas P. vulgaris accounted for the least, 7/285 (2.5%). Overall, 132/285 (46.3%) and 99/285 (34.7%) of culture-positive patients were infected by extended-spectrum beta-lactamase and carbapenemase-producing bacteria. The overall multidrug resistance rate of the isolated bacteria was 89.4%. The highest antibiotic resistance rates were detected for doxycycline (92.9%), amoxicillin-clavulanic acid (83.9%), and ampicillin (93%). The least antibiotic resistance rate was observed for meropenem at 41.1% and amikacin at 1.7%, respectively. CONCLUSIONS AND RECOMMENDATIONS: In the study area, significant health concerns include a range of hospital-acquired bacterial infections associated with elevated rates of multidrug resistance, Extended-spectrum beta-lactamase (ESBL), and carbapenemase-producing bacterial pathogens. Consequently, it is recommended to conduct drug-susceptibility testing of isolates and molecular detection at a national level to optimize antibiotic usage for treating prevalent bacterial infections in this area.

Asunto(s)

Antibacterianos , Infección Hospitalaria , Farmacorresistencia Bacteriana Múltiple , Bacterias Gramnegativas , Infecciones por Bacterias Gramnegativas , Pruebas de Sensibilidad Microbiana , Humanos , Etiopía/epidemiología , Estudios Transversales , Masculino , Femenino , Adulto , Persona de Mediana Edad , Antibacterianos/farmacología , Infección Hospitalaria/microbiología , Infección Hospitalaria/epidemiología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/aislamiento & purificación , Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/genética , Adulto Joven , Adolescente , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/epidemiología , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Farmacorresistencia Bacteriana Múltiple/genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Anciano , Niño , Preescolar , Lactante , Hospitalización/estadística & datos numéricos , Proteínas Bacterianas/genética , Anciano de 80 o más Años

RESUMEN

BACKGROUND: Extended-spectrum ß-lactamase -producing Enterobacterales (ESBL-E) are important zoonotic pathogens that can cause serious clinical infections, also in horses. Preventing the spread of ESBL-E, especially in the equine hospital environment, is key to reducing the number of difficult-to-treat infections. Estimating the local prevalence of ESBL-E in horses is crucial to establish targeted infection control programs at equine hospitals. We conducted a prevalence and risk factor study in equine patients on admission to an equine teaching hospital in Finland through a rectal ESBL-E screening specimen of the horse and a questionnaire. RESULTS: The prevalence of ESBL-E in admitted horses was 3% (5/161, 95% CI 1-7%); none of the tested factors remained statistically significant in multivariate analysis, although antimicrobial treatment within three months was borderline significant (p = 0.052). Extended-spectrum ß-lactamase -producing Klebsiella pneumoniae ST6179:CTX-M-15 was detected in three horses using whole-genome sequencing, which in combination with patient records suggested nosocomial transmission. Escherichia coli isolates were ST1250:CTX-M-1 (n = 1), ST1079:CTX-M-1 (n = 1), and ST1245:CTX-M-14 (n = 1). Multiple virulence genes were detected in the ESBL-E isolates. In the ESBL-E positive horses enrolled in a one-year follow-up study, ESBL-E were unlikely to be isolated in rectal screening specimens after the initial positive specimen. CONCLUSIONS: The prevalence of ESBL-E in horses visiting a veterinary teaching hospital in Finland is low, indicating an overall low prevalence estimate in the country's equine population. No statistically significant risk factors were identified, likely due to the low number of cases. The duration of ESBL-E carriage is likely to be very short in horses.

Asunto(s)

Infecciones por Enterobacteriaceae , Enfermedades de los Caballos , Hospitales Veterinarios , beta-Lactamasas , Animales , Caballos , Enfermedades de los Caballos/microbiología , Enfermedades de los Caballos/epidemiología , beta-Lactamasas/metabolismo , beta-Lactamasas/genética , Prevalencia , Factores de Riesgo , Finlandia/epidemiología , Infecciones por Enterobacteriaceae/veterinaria , Infecciones por Enterobacteriaceae/epidemiología , Infecciones por Enterobacteriaceae/microbiología , Masculino , Femenino , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/aislamiento & purificación , Infección Hospitalaria/epidemiología , Infección Hospitalaria/veterinaria , Infección Hospitalaria/microbiología , Escherichia coli/enzimología , Escherichia coli/aislamiento & purificación , Enterobacteriaceae/enzimología , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/efectos de los fármacos , Antibacterianos/farmacología

RESUMEN

Fosfomycin (FOS) is an effective antibiotic against multidrug-resistant Enterobacterales, but its effectiveness is reducing. Little is known on the current prevalence of FosA enzymes in low-risk pathogens, such as Citrobacter freundii. The aim of the study was the molecular characterization of a carbapenemase- and FosA-producing C. freundii collected in Italy. AK867, collected in 2023, showed an XDR profile, retaining susceptibility only to colistin. AK867 showed a FOS MIC >128 mg/L by ADM. Based on WGS, AK867 belonged to ST116 and owned a wide resistome, including fosA3, blaKPC-2, and blaVIM-1. fosA3 was carried by a conjugative pKPC-CAV1312 plasmid of 320,480 bp, on a novel composite transposon (12,907 bp). FosA3 transposon shared similarities with other fosA3-harboring pKPC-CAV1312 plasmids among Citrobacter spp. We report the first case of FosA3 production in clinical carbapenemase-producing C. freundii ST116. The incidence of FosA3 enzymes is increasing among Enterobacterales, affecting even low-virulence pathogens, as C. freundii.

Asunto(s)

Antibacterianos , Proteínas Bacterianas , Citrobacter freundii , Infecciones por Enterobacteriaceae , Fosfomicina , beta-Lactamasas , Humanos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Citrobacter freundii/genética , Citrobacter freundii/enzimología , Citrobacter freundii/efectos de los fármacos , Elementos Transponibles de ADN , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Enterobacteriaceae/microbiología , Fosfomicina/farmacología , Italia/epidemiología , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Secuenciación Completa del Genoma

RESUMEN

BACKGROUND: Urolithiasis combined with ESBL-producing E. coli is often difficult to control and leads to higher postoperative infection-related complications. This study was aim to explore the efficacy and necessity for early use of carbapenem antibiotics perioperatively in urolithiasis patients with urinary tract infections caused by ESBL-producing E. coli. METHODS: The study included a total of 626 patients who were separated into two groups: Group I (the ESBL-producing E. coli group) and Group II (the non-ESBL-producing E. coli group). Antibiotic susceptibility testing was performed and the two groups induced postoperative infection-related events were recorded. the efficacy of perioperative antibiotics was evaluated. RESULTS: All strains of E. coli in our research were sensitive to Carbapenems antibiotics. In addition to Carbapenems, the resistance rates of ESBL-producing E. coli to 6 other commonly used antibiotics were higher than those of non-ESBL-producing strains. Based on the preoperative antibiotic susceptibility test for the ESBL-producing E. coli group and the qSOFA score, the Carbapenems were more effective than the ß-lactamase inhibitors (p = 0.08), while for the non-ESBL-producing E. coli group, there was no difference in the treatment effects between Carbapenems, ß-lactamase inhibitors, Ceftazidime and Quinolones (p = 0.975). CONCLUSIONS: Carbapenem antibiotics significantly reduced the incidence of postoperative infection-related events compared with other types of antibiotics for ESBL-producing E. coli infections in patient with urolithiasis.

Asunto(s)

Carbapenémicos , Infecciones por Escherichia coli , Escherichia coli , Urolitiasis , beta-Lactamasas , Humanos , Carbapenémicos/uso terapéutico , Escherichia coli/efectos de los fármacos , Urolitiasis/tratamiento farmacológico , Femenino , Masculino , Persona de Mediana Edad , beta-Lactamasas/metabolismo , Infecciones por Escherichia coli/tratamiento farmacológico , Anciano , Antibacterianos/uso terapéutico , Infecciones Urinarias/tratamiento farmacológico , Atención Perioperativa , Adulto , Estudios Retrospectivos , Pruebas de Sensibilidad Microbiana , Resultado del Tratamiento

RESUMEN

The global proliferation of carbapenemase-producing bacteria (CPB) has garnered significant attention worldwide. Early diagnosis of CPB and accurate identification of carbapenemases are crucial for preventing the spread of CPB and ensuring targeted antibiotic therapy. Therefore, efficient and accurate identification of carbapenemases is paramount in clinically treating diseases associated with CPB. In this study, 58 CPB strains were collected and detected using the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) method, a rapid detection platform based on CRISPR-Cas12a gene editing and isothermal amplification. Additionally, four conventional methods (the APB/EDTA method, PCR, NG-test Carba 5, and GeneXpert Carba-R) were employed and compared against whole genome sequencing (WGS) results, considered the gold standard, to evaluate their efficacy in detecting carbapenemases. Detection by the APB/EDTA method revealed that 29 strains were positive for Class A serine endopeptidases, while 29 strains were positive for Class B metalloenzymes. The classification of these zymotypes was consistent with the sequencing result. All target carbapenemases for KPC were identified with 100% sensitivity using NG-test Carba 5, PCR, DETECTR, and GeneXpert Carba-R. In the case of NDM, both Xpert Carba-R and DETECTR showed a sensitivity of 100%. In contrast, NG-test Carba 5 and PCR had a slightly lower sensitivity of 96.7%, each missing one target carbapenemase. n this study, the APB/EDTA method is capable of identifying the zymotype classification but not the specific resistant genes, while Xpert Carba-R and DETECTR are able to detect all target carbapenemases.

Asunto(s)

Proteínas Bacterianas , beta-Lactamasas , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Sensibilidad y Especificidad , Reacción en Cadena de la Polimerasa/métodos , Secuenciación Completa del Genoma , Sistemas CRISPR-Cas

RESUMEN

Antibiotic resistance poses a persistent threat to modern medicine due to the emergence of novel antibiotic-resistant strains. Therefore, a timely understanding of antibiotic resistance and the virulence biology of pathogenic bacteria, particularly those of public health significance, is crucial for implementing effective mitigation strategies. This study aimed to investigate the virulence profiles of ten S. aureus isolates (NDa to NDj) and ten E. coli isolates (ND1 to ND10) originating from livestock and poultry, and to assess how various cell surface properties and biofilm formation abilities influence antibiotic resistance phenotypes. Antibiotic resistance profiling through phenotypic (AST) and genotypic methods (PCR) confirmed that NDa to NDe were methicillin-resistant S. aureus (MRSA) and ND1 to ND5 were extended-spectrum ß-lactamase (ESBL) producing E. coli isolates. Virulence properties such as hemolytic activity, coagulase activity, and nuclease activity were found to be independent of the antibiotic resistance phenotype in S. aureus. In contrast, biofilm formation phenotype was observed to influence antibiotic resistance phenotypes, with MRSA and ESBL E. coli isolates demonstrating higher biofilm formation potency. Chemical and enzymatic analysis of S. aureus and E. coli biofilms revealed proteins and polysaccharides as major components, followed by nucleic acids. Furthermore, cell surface properties such as auto-aggregation and hydrophobicity were notably higher in isolates with strong to medium biofilm-forming capabilities (ESBL and MRSA isolates), corroborated by genomic confirmation of various genes associated with biofilm, adhesion, and colonization. In conclusion, this study highlights that surface hydrophobicity and biofilm formation ability of MRSA (NDa to NDe) and ESBL E. coli (ND1 to ND5) isolates may influence antibiotic resistance phenotypes.

Asunto(s)

Antibacterianos , Biopelículas , Escherichia coli , Ganado , Staphylococcus aureus Resistente a Meticilina , Pruebas de Sensibilidad Microbiana , Aves de Corral , Factores de Virulencia , beta-Lactamasas , Biopelículas/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Animales , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/patogenicidad , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Staphylococcus aureus Resistente a Meticilina/enzimología , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Aves de Corral/microbiología , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Ganado/microbiología , Virulencia , Antibacterianos/farmacología , Propiedades de Superficie , Genotipo , Fenotipo , Infecciones Estafilocócicas/microbiología

RESUMEN

Objective: This study aimed to comprehensively investigate hypervirulent carbapenem-resistant Klebsiella pneumoniae (CR-hvKP) in the Ningbo region. Importantly, we sought to elucidate its molecular characteristics and pathogenic mechanisms. This information will provide evidence-based insights for preventing and controlling nosocomial infections and facilitate improved clinical diagnosis and treatment in this region. Methods: 96 carbapenem-resistant Klebsiella pneumoniae strains were collected from the Ningbo region between January 2021 and December 2022. Whole genome sequencing and bioinformatic methods were employed to identify and characterize CR-hvKP strains at the molecular level. The minimum inhibitory concentrations (MICs) of common clinical antibiotics were determined using the VITEK-2 Compact automatic microbiological analyzer. Plasmid conjugation experiments evaluated the transferability of resistance plasmids. Finally, mouse virulence assays were conducted to explore the pathogenic mechanisms. Results: Among the 96 strains, a single CR-hvKP strain, designated CR-hvKP57, was identified, with an isolation frequency of 1.04%. Whole-genome sequencing revealed the strain to be ST23 serotype with a K1 capsule. This strain harbored three plasmids. Plasmid 1, a pLVPK-like virulence plasmid, carried multiple virulence genes, including rmpA, rmpA2, iroB, iucA, and terB. Plasmid 2 contained transposable element sequences such as IS15 and IS26. Plasmid 3, classified as a resistance plasmid, harbored the bla KPC-3 carbapenem resistance gene. Mouse virulence assays demonstrated a high mortality rate associated with CR-hvKP57 infection. Additionally, there was a significant increase in IL-1ß, IL-6, and TNF-α levels in response to CR-hvKP57 infection, indicating varying degrees of inflammatory response. Western blot experiments further suggested that the pathogenic mechanism involves activation of the NF-κB signaling pathway. Conclusion: This study confirms the emergence of hypervirulent CR-hvKP in the Ningbo region, which likely resulted from the acquisition of a pLVPK-like virulence plasmid and a bla KPC-3 resistance plasmid by the ST23-K1 type Klebsiella pneumoniae. Our findings highlight the urgent need for more judicious use of antibiotics to limit the emergence of resistance. Additionally, strengthening infection prevention and control measures is crucial to minimize the spread of virulence and resistance plasmids.

Asunto(s)

Antibacterianos , Proteínas Bacterianas , Enterobacteriaceae Resistentes a los Carbapenémicos , Carbapenémicos , Infecciones por Klebsiella , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , Plásmidos , Secuenciación Completa del Genoma , beta-Lactamasas , Animales , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidad , Klebsiella pneumoniae/efectos de los fármacos , Infecciones por Klebsiella/microbiología , Ratones , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Plásmidos/genética , Virulencia/genética , Humanos , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Enterobacteriaceae Resistentes a los Carbapenémicos/patogenicidad , Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbapenémicos/farmacología , China , Factores de Virulencia/genética , Femenino , Modelos Animales de Enfermedad , Masculino

RESUMEN

Antibiotic resistance is one of most important health concerns nowadays, and ß-lactamases are the most important resistance determinants. These enzymes, based on their structural and functional characteristics, are grouped in four categories (A, B, C and D). We have solved the structure of PIB-1, a Pseudomonas aeruginosa chromosomally-encoded ß-lactamase, in its apo form and in complex with meropenem and zinc. These crystal structures show that it belongs to the Class C ß-lactamase group, although it shows notable differences, especially in the Ω- and P2-loops, which are important for the enzymatic activity. Functional analysis showed that PIB-1 is able to degrade carbapenems but not cephalosporins, the typical substrate of Class C ß-lactamases, and that its catalytic activity increases in the presence of metal ions, especially zinc. They do not bind to the active-site but they induce the formation of trimers that show an increased capacity for the degradation of the antibiotics, suggesting that this oligomer is more active than the other oligomeric species. While PIB-1 is structurally a Class C ß-lactamase, the low sequence conservation, substrate profile and its metal-dependence, prompts us to position this enzyme as the founder of a new group inside the Class C ß-lactamases. Consequently, its diversity might be wider than expected.

Asunto(s)

Carbapenémicos , Pseudomonas aeruginosa , Zinc , beta-Lactamasas , Pseudomonas aeruginosa/enzimología , beta-Lactamasas/química , beta-Lactamasas/metabolismo , Carbapenémicos/farmacología , Carbapenémicos/metabolismo , Carbapenémicos/química , Zinc/metabolismo , Zinc/química , Modelos Moleculares , Dominio Catalítico , Hidrólisis , Especificidad por Sustrato , Metales/metabolismo , Metales/química , Metales/farmacología , Relación Estructura-Actividad , Meropenem/farmacología , Meropenem/química , Meropenem/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X

RESUMEN

Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum ß-lactam antibiotics in Gram-negative bacteria is a major impediment to treating infections. Genes responsible for antibiotic resistance are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the conjugation of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based flow cytometry assay, we found that the natural products were more active in reducing transmission of the IncK extended-spectrum ß-lactamase-encoding plasmid pCT in Escherichia coli EC958c, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII carbapenemase-encoding plasmid pKpQIL. The exception was the natural product rottlerin, also active in K. pneumoniae. In classical conjugation assays, rottlerin also reduced the conjugation frequency of the IncFII bla NDM-1 carrying plasmid pCPE16_3 from a clinical K. pneumoniae isolate. Our data indicate that the natural products tested here, in their current molecular structure, reduced conjugation by a small amount, which is unlikely to achieve a large-scale reduction in AMR in bacterial populations. However, certain natural products like rottlerin could provide a foundation for further research into compounds with effective anti-plasmid activity.

Asunto(s)

Antibacterianos , Productos Biológicos , Escherichia coli , Klebsiella pneumoniae , Plásmidos , beta-Lactamasas , Plásmidos/genética , Antibacterianos/farmacología , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Productos Biológicos/farmacología , Farmacorresistencia Bacteriana/genética , Conjugación Genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/genética , Pruebas de Sensibilidad Microbiana , Microbiología de Alimentos , Transferencia de Gen Horizontal

RESUMEN

Introduction: Extended-spectrum ß-lactamase (ESBL) production among Enterobacteriaceae, such as E. coli, has been increasing worldwide, which causes treatment failure for urinary tract infections. Therefore, this study aimed to determine the prevalence and risk factors for the production of ESBL in E. coli from patients with urinary tract infections (UTI) in Zanzibar. Methods: a prospective cross-sectional study was conducted from January 2018 to December 2021 in Zanzibar. Data were retrieved from a routine bacteriological laboratory culture report from urine samples of 4306 patients at the Lancet Laboratory. In addition, the patient's social demographics and clinical data were retrieved by examining the medical records in the respective hospitals. All inpatients older than fifteen years diagnosed with urinary tract infections (UTI) and requested urine culture and sensitivity were included. The Chi-square and Fischer's exact tests were used to compare antibiotic resistance. In addition, a binary logistic regression analysis was used to predict ESBL production risk factors. Results: the prevalence of E. coli-producing ESBL was 13.4% (578/4030). Infection of ESBL. E. coli was prevalent in females 52.6% (n=304) compared to male patients, 47.4% (n=274), and the majority 38.8% (n=224), were people of young age, between 16-30 years. The average age of patients was 31.5±10.2 years, with minimum age of 16 years and a maximum age of 72 years. In multivariate analysis, results shown that previously hospitalised patients aOR: 6.35, 95% Cl 3.37-11.92; p=0.001, long hospital stays aOR: 10.34, 95% Cl 3.03-22.29; p <0.001, prior use of penicillin aOR: 7.78, 95% Cl 2.99-29.11; p < 0.001, and prior use of cephalosporin drugs aOR: 4.64, 95% Cl 2.99-9.96; p=0.001, were strongly associated with the emergence of ESBL-producing E. coli in urinary tract infection patients. ESBL E. coli showed high resistance to amoxicillin 99.5% (n=575), ampicillin 97.8.% (n=570), cotrimazaxole 86.2% (n=344), ceftriaxone 73.7% (n=344), ciprofloxacin 73.2% (n=423), and ceftaxime 59.5% (n=426). There was a less resistance to ampicillin -cloxacillin 44.3% (n=256), gentamicin 22.5% (n=22.5), and norfloxacin 18.9% (n=109) respectively. Isolates were shown to be more susceptible to meropenem at 1.6% (n=9). Conclusion: the overall prevalence of ESBL-producing E. coli is 13.4%. The risk of emergence ESBL was higher in patients with previous history of hospitalisation, long hospital stay, prior use of penicillin and cephalosporin drugs. High level of antimicrobial resistance observed against most commonly used antibiotics in treatment of urinary tract infections. The clinicians should rely on microbiological diagnosis in treatment of UTIs to reduce risk of treatment failure. Further study should be carried out to assess the prevalence and resistance pattern of other uropathogens and other risk factors.

Asunto(s)

Antibacterianos , Infecciones por Escherichia coli , Escherichia coli , Centros de Atención Terciaria , Infecciones Urinarias , beta-Lactamasas , Humanos , Infecciones Urinarias/microbiología , Infecciones Urinarias/epidemiología , Infecciones Urinarias/tratamiento farmacológico , Estudios Transversales , Femenino , Estudios Prospectivos , Masculino , Factores de Riesgo , beta-Lactamasas/metabolismo , Escherichia coli/aislamiento & purificación , Escherichia coli/efectos de los fármacos , Prevalencia , Adulto , Persona de Mediana Edad , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Antibacterianos/farmacología , Adulto Joven , Tanzanía/epidemiología , Anciano , Adolescente , Farmacorresistencia Bacteriana , Pacientes Internos/estadística & datos numéricos , Pruebas de Sensibilidad Microbiana

RESUMEN

The extended-spectrum ß-lactamases (ESßLs) are bacterial enzymes capable of hydrolyzing penicillins, cephalosporins, and aztreonam. The prevalence of ESßL is increasing among clinically significant microorganisms worldwide, drastically reducing the therapeutic management of infectious diseases. The study aimed to determine the drug susceptibility of ESßL-positive clinical isolates acquired from patients hospitalized in Lodz, central Poland, and analyze the prevalence of specific genes, determining acquired resistance in these bacteria. The samples of ESßL-positive clinical isolates were gathered in 2022 from medical microbiological laboratories in the city of Lodz, central Poland. The strains were subjected to biochemical identification and antimicrobial susceptibility testing following EUCAST guidelines. The presence of studied genes (blaCTX-M, blaSHV, blaTEM, blaPER, blaVEB) was confirmed by PCR. Over 50% of studied isolates were resistant to gentamicin, cefepime, ceftazidime and ciprofloxacin. The most common ESßL gene was blaCTX-M. In most isolates, the resistance genes occurred simultaneously. The blaPER was not detected in any of the tested strains. ESßL-producing strains are largely susceptible to the currently available antibiotics. The observation of the coexistence of different genes in most clinical isolates is alarming.

Asunto(s)

Antibacterianos , Infecciones por Enterobacteriaceae , Enterobacteriaceae , Pruebas de Sensibilidad Microbiana , beta-Lactamasas , Humanos , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Polonia/epidemiología , Antibacterianos/farmacología , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/epidemiología , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Enterobacteriaceae/genética , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/enzimología , Epidemiología Molecular , Masculino , Femenino , Adulto , Persona de Mediana Edad , Ciprofloxacina/farmacología

RESUMEN

BACKGROUND: In the hospital environment, carbapenemase-producing Pseudomonas aeruginosa (CPPA) may lead to fatal patient infections. However, the transmission routes of CPPA often remain unknown. Therefore, this case study aimed to trace the origin of CPPA ST357, which caused a hospital-acquired pneumonia in a repatriated critically ill patient suffering from Guillain-Barré Syndrome in 2023. METHODS: Antimicrobial susceptibility of the CPPA isolate for 30 single and combination therapies was determined by disk-diffusion, Etest or broth microdilution. Whole-genome sequencing was performed for three case CPPA isolates (one patient and two sinks) and four distinct CPPA ST357 patient isolates received in the Dutch CPPA surveillance program. Furthermore, 193 international P. aeruginosa ST357 assemblies were collected via three genome repositories and analyzed using whole-genome multi-locus sequence typing in combination with antimicrobial resistance gene (ARG) characterization. RESULTS: A Dutch patient who carried NDM-1-producing CPPA was transferred from Kenya to the Netherlands, with subsequent dissemination of CPPA isolates to the local sinks within a month after admission. The CPPA case isolates presented an extensively drug-resistant phenotype, with susceptibility only for colistin and cefiderocol-fosfomycin. Phylogenetic analysis showed considerable variation in allelic distances (mean = 150, max = 527 alleles) among the ST357 isolates from Asia (n = 92), Europe (n = 58), Africa (n = 21), America (n = 16), Oceania (n = 2) and unregistered regions (n = 4). However, the case isolates (n = 3) and additional Dutch patient surveillance program isolates (n = 2) were located in a sub-clade of isolates from Kenya (n = 17; varying 15-49 alleles), the United States (n = 7; 21-115 alleles) and other countries (n = 6; 14-121 alleles). This was consistent with previous hospitalization in Kenya of 2/3 Dutch patients. Additionally, over half of the isolates (20/35) in this sub-clade presented an identical resistome with 9/17 Kenyan, 5/5 Dutch, 4/7 United States and 2/6 other countries, which were characterized by the blaNDM-1, aph(3')-VI, ARR-3 and cmlA1 ARGs. CONCLUSION: This study presents an extensively-drug resistant subclone of NDM-producing P. aeruginosa ST357 with a unique resistome which was introduced to the Netherlands via repatriation of critically ill patients from Kenya. Therefore, the monitoring of repatriated patients for CPPA in conjunction with vigilance for the risk of environmental contamination is advisable to detect and prevent further dissemination.

Asunto(s)

Antibacterianos , Farmacorresistencia Bacteriana Múltiple , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Secuenciación Completa del Genoma , beta-Lactamasas , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/enzimología , Países Bajos/epidemiología , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/epidemiología , Infecciones por Pseudomonas/tratamiento farmacológico , Farmacorresistencia Bacteriana Múltiple/genética , Antibacterianos/farmacología , Kenia/epidemiología , Tipificación de Secuencias Multilocus , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Masculino

RESUMEN

The effectiveness of ß-lactam antibiotics is increasingly threatened by resistant bacteria that harbor hydrolytic ß-lactamase enzymes. Depending on the class of ß-lactamase present, ß-lactam hydrolysis can occur through one of two general molecular mechanisms. Metallo-ß-lactamases (MBLs) require active site Zn2+ ions, whereas serine-ß-lactamases (SBLs) deploy a catalytic serine residue. The result in both cases is drug inactivation via the opening of the ß-lactam warhead of the antibiotic. MBLs confer resistance to most ß-lactams and are non-susceptible to SBL inhibitors, including recently approved diazabicyclooctanes, such as avibactam; consequently, these enzymes represent a growing threat to public health. Aspergillomarasmine A (AMA), a fungal natural product, can rescue the activity of the ß-lactam antibiotic meropenem against MBL-expressing bacterial strains. However, the effectiveness of this ß-lactam/ß-lactamase inhibitor combination against bacteria producing multiple ß-lactamases remains unknown. We systematically investigated the efficacy of AMA/meropenem combination therapy with and without avibactam against 10 Escherichia coli and 10 Klebsiella pneumoniae laboratory strains tandemly expressing single MBL and SBL enzymes. Cell-based assays demonstrated that laboratory strains producing NDM-1 and KPC-2 carbapenemases were resistant to the AMA/meropenem combination but became drug-susceptible upon adding avibactam. We also probed these combinations against 30 clinical isolates expressing multiple ß-lactamases. E. coli, Enterobacter cloacae, and K. pneumoniae clinical isolates were more susceptible to AMA, avibactam, and meropenem than Pseudomonas aeruginosa and Acinetobacter baumannii isolates. Overall, the results demonstrate that a triple combination of AMA/avibactam/meropenem has potential for empirical treatment of infections caused by multiple ß-lactamase-producing bacteria, especially Enterobacterales.

Asunto(s)

Antibacterianos , Compuestos de Azabiciclo , Escherichia coli , Meropenem , Pruebas de Sensibilidad Microbiana , beta-Lactamasas , Compuestos de Azabiciclo/farmacología , beta-Lactamasas/metabolismo , beta-Lactamasas/genética , Antibacterianos/farmacología , Meropenem/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/enzimología , Inhibidores de beta-Lactamasas/farmacología , Humanos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/genética , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Combinación de Medicamentos , Enterobacter cloacae/efectos de los fármacos , Enterobacter cloacae/enzimología , Ácido Aspártico/análogos & derivados

RESUMEN

Taniborbactam, a bicyclic boronate ß-lactamase inhibitor with activity against Klebsiella pneumoniae carbapenemase (KPC), Verona integron-encoded metallo-ß-lactamase (VIM), New Delhi metallo-ß-lactamase (NDM), extended-spectrum beta-lactamases (ESBLs), OXA-48, and AmpC ß-lactamases, is under clinical development in combination with cefepime. Susceptibility of 200 previously characterized carbapenem-resistant K. pneumoniae and 197 multidrug-resistant (MDR) Pseudomonas aeruginosa to cefepime-taniborbactam and comparators was determined by broth microdilution. For K. pneumoniae (192 KPC; 7 OXA-48-related), MIC90 values of ß-lactam components for cefepime-taniborbactam, ceftazidime-avibactam, and meropenem-vaborbactam were 2, 2, and 1 mg/L, respectively. For cefepime-taniborbactam, 100% and 99.5% of isolates of K. pneumoniae were inhibited at ≤16 mg/L and ≤8 mg/L, respectively, while 98.0% and 95.5% of isolates were susceptible to ceftazidime-avibactam and meropenem-vaborbactam, respectively. For P. aeruginosa, MIC90 values of ß-lactam components of cefepime-taniborbactam, ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam were 16, >8, >8, and >4 mg/L, respectively. Of 89 carbapenem-susceptible isolates, 100% were susceptible to ceftolozane-tazobactam, ceftazidime-avibactam, and cefepime-taniborbactam at ≤8 mg/L. Of 73 carbapenem-intermediate/resistant P. aeruginosa isolates without carbapenemases, 87.7% were susceptible to ceftolozane-tazobactam, 79.5% to ceftazidime-avibactam, and 95.9% and 83.6% to cefepime-taniborbactam at ≤16 mg/L and ≤8 mg/L, respectively. Cefepime-taniborbactam at ≤16 mg/L and ≤8 mg/L, respectively, was active against 73.3% and 46.7% of 15 VIM- and 60.0% and 35.0% of 20 KPC-producing P. aeruginosa isolates. Of all 108 carbapenem-intermediate/resistant P. aeruginosa isolates, cefepime-taniborbactam was active against 86.1% and 69.4% at ≤16 mg/L and ≤8 mg/L, respectively, compared to 59.3% for ceftolozane-tazobactam and 63.0% for ceftazidime-avibactam. Cefepime-taniborbactam had in vitro activity comparable to ceftazidime-avibactam and greater than meropenem-vaborbactam against carbapenem-resistant K. pneumoniae and carbapenem-intermediate/resistant MDR P. aeruginosa.

Asunto(s)

Antibacterianos , Cefepima , Farmacorresistencia Bacteriana Múltiple , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa , Inhibidores de beta-Lactamasas , Cefepima/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Klebsiella pneumoniae/efectos de los fármacos , Antibacterianos/farmacología , Inhibidores de beta-Lactamasas/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Cefalosporinas/farmacología , Humanos , beta-Lactamasas/metabolismo , beta-Lactamasas/genética , Ácidos Borónicos/farmacología , Carbapenémicos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ceftazidima/farmacología , Ácidos Borínicos/farmacología , Combinación de Medicamentos , Compuestos de Azabiciclo/farmacología , Ácidos Carboxílicos

RESUMEN

Aim -To isolate bacteriophages targeting extended-spectrum beta-lactamase-producing K. pneumoniae and evaluate their effectiveness across diverse models, incorporating innovative alternatives in animal testing. METHODS AND RESULTS: vB_kpnS-Kpn15 was isolated from sewage sample from Thane district. It produced a clear plaques on K. pneumoniae ATCC 700603. It has a flexible, non-contractile long tail and an icosahedral head and the Siphoviridae family of viruses in the order Caudovirales matched all of its structural criteria. Sequencing of vB_kpnS-Kpn15 revealed a 48,404 bp genome. The vB_KpnS-Kpn15 genome was found to contain 50 hypothetical proteins, of which 16 were found to possess different functions. The vB_KpnS-Kpn15 was also found to possess enzymes for its DNA synthesis. It was found to be lytic for the planktonic cells of K. pneumoniae and bactericidal for up to 48 h and potentially affected established K. pneumoniae biofilms. It demonstrated a broad host range and caused lytic zones on about 46 % of K. pneumoniae multi-drug resistant strains. In an in vitro wound and burn infection model, phage vB_kpnS-Kpn15 in combination with other phages resulted in successful cell proliferation and wound healing. Based on vB_kpnS-Kpn15's lytic properties, it can be incorporated in a bacteriophage cocktail to combat ESBL strains. CONCLUSIONS: The phages isolated during this research are better candidates for phage therapy, and therefore provide new and exciting options for the successful control of antibiotic-resistant bacterial infections in the future. The utilization of animal alternative models in this study elucidates cellular proliferation and migration, underscoring its significance in screening novel drugs with potential applications in the treatment of wound and burn infections. SIGNIFICANCE AND IMPACT OF THE RESEARCH: The findings of this research have implications for the creation of innovative, promising strategies to treat ESBL K. pneumoniae infections.

Asunto(s)

Bacteriófagos , Biopelículas , Modelos Animales de Enfermedad , Genoma Viral , Especificidad del Huésped , Infecciones por Klebsiella , Klebsiella pneumoniae , Terapia de Fagos , Aguas del Alcantarillado , beta-Lactamasas , Klebsiella pneumoniae/virología , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Animales , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/terapia , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Biopelículas/crecimiento & desarrollo , Aguas del Alcantarillado/microbiología , Aguas del Alcantarillado/virología , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple , Humanos , Ratones , Infección de Heridas/microbiología , Infección de Heridas/terapia , Caudovirales/genética , Caudovirales/aislamiento & purificación , Siphoviridae/genética , Siphoviridae/aislamiento & purificación , Siphoviridae/fisiología , Pruebas de Sensibilidad Microbiana

RESUMEN

A novel KPC variant, KPC-84, identified in a Klebsiella pneumoniae isolate from China, exhibits a threonine (T) to proline (P) amino acid substitution at Ambler position 243(T243P), altering from the KPC-2 sequence. Cloning and expression of blaKPC-84 in Escherichia coli, with subsequent MIC assessments, revealed increased resistance to ceftazidime-avibactam and significantly reduced carbapenemase activity compared to KPC-2. Kinetic measurements showed that KPC-84 exhibited sligthly higher hydrolysis of ceftazidime and reduced affinity for avibactam compared to KPC-2. This study emphasizes the emerging diversity of KPC variants with ceftazidime-avibactam resistance, underscoring the complexity of addressing carbapenem-resistant Klebsiella pneumoniae infections.

Asunto(s)

Antibacterianos , Compuestos de Azabiciclo , Proteínas Bacterianas , Ceftazidima , Combinación de Medicamentos , Infecciones por Klebsiella , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , beta-Lactamasas , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/aislamiento & purificación , Compuestos de Azabiciclo/farmacología , Ceftazidima/farmacología , Humanos , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/tratamiento farmacológico , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , China , Sustitución de Aminoácidos , Escherichia coli/genética , Escherichia coli/efectos de los fármacos

RESUMEN

ß-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum ß-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine ß­lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential ß-lactamase inhibitor.In the present study, blaCTX-M-15 and blaKPC-2 represented genes encoding for serine ß-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64-128 µg/mL) and cephalosporins (MICs; 128-512 µg/mL). The MICs of the tested ß-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-ß-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64-512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a ß-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC50 values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-ß-lactam inhibitor, which interacts with the crucial residues involved in ß-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme's active site. GQA markedly enhanced the potency of ß-lactams against carbapenemase and extended-spectrum ß-lactamase-producing strains, reducing the MICs of ß-lactams to the susceptible range. The ß-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent ß-lactamase inhibitors.

Asunto(s)

Escherichia coli , Pruebas de Sensibilidad Microbiana , Inhibidores de beta-Lactamasas , beta-Lactamasas , beta-Lactamasas/metabolismo , beta-Lactamasas/genética , Inhibidores de beta-Lactamasas/farmacología , Escherichia coli/genética , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/genética , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/antagonistas & inhibidores , Carbapenémicos/farmacología

RESUMEN

Staphylococcus aureus is a bacterial pathogen that causes bloodstream infections, pneumonia, and skin abscesses and is the primary pathogen responsible for medical devices associated with biofilm infections, accounting for approximately 70 % of cases. Therefore, the World Health Organization (WHO) has designated this microorganism as a top priority due to its role in causing over 20,000 bacteremia-related deaths in the US each year. The issue of pathogen resistance to antibiotics, mainly by a biofilm, further complicates these infections since biofilms render the bacterial colony impervious to antibiotics. However, many natural and synthetic substances also induce bacterial biofilm formation. Therefore, we investigated whether the most common active pharmaceutical ingredients (APIs) could induce biofilm formation in two clinical isolates of extended-spectrum beta-lactamase Staphylococcus aureus, one of them also methicillin-resistant (A2M) and two medical devices. We detected biofilm inducers, inhibitors, and destabilizers. Microbial strain, medical devices, API structure, and concentration influenced the modulatory effects of biofilm. In all devices tested, including microplates, FR18 duodenal probe, and respiratory probe, the clinic isolate methicillin-resistant S. aureus A2M exhibited lower susceptibility to biofilm formation than S. aureus A1. The anti-inflammatory acetaminophen, the hypocholesterolemic lovastatin, and the diuretic hydrochlorothiazide all induced biofilm. However, verapamil, an antihypertensive, and cetirizine, an antihistamine, inhibited biofilm on S. aureus A2M, while propranolol, another antihypertensive, inhibited biofilm on S. aureus A1. Additionally, diclofenac, an analgesic, and cetirizine destabilized the biofilm, resulting in more free bacteria and possibly making them more susceptible to external agents such as antibiotics. Nonetheless, further epidemiologic analyses and in vivo assays are needed to confirm these findings and to establish a correlation between drug use, the onset of bacterial infections in patients, and the use of medical devices. This work provides information about the probable clinical implications of drugs in patients using medical devices or undergoing surgical procedures. Inhibitory APIs could also be used as drug repurposing or templates to design new, more potent biofilm inhibitors.

Asunto(s)

Antibacterianos , Biopelículas , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas , Staphylococcus aureus , beta-Lactamasas , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Antibacterianos/farmacología , Humanos , Staphylococcus aureus/efectos de los fármacos , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , beta-Lactamasas/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos

RESUMEN

ESBL-producing Escherichia coli strains threaten public health and obligate the use of last-resort antibiotics. This study identified 15 E. coli isolates through 16S rRNA and gyrB genes, specific to E. coli, in 120 egg samples (12.5%). Antibiotic resistance was detected according to the EUCAST and CLSI in E. coli isolates. 2 isolates were susceptible to all antibiotics, one isolate was resistant to one antibiotic, one isolate was resistant to 2 antibiotics, and 11 E. coli isolates (73.3%) had multidrug resistance. Most frequent antibiotic resistances were detected against ampicillin (80%), tetracycline (66.6%), and chloramphenicol (66.6%). A double-disc confirmation test was used to detect ESBL production, and blaTEM, blaSHV, blaCTX-M and blaOXA genes were searched by PCR. The blaTEM (100%) gene was found in all resistant E. coli isolates, and the blaCTX-M gene was detected in only 3 (20%) E. coli isolates. None of the E. coli isolates contained the genes responsible for carbapenem and colistin resistance. Our results show that multi-drug antibiotic resistance and the blaTEM gene are frequent in E. coli from table eggs in Istanbul. This is the first preliminary study on ESBL-producing E. coli isolates in table eggs in Türkiye.

Asunto(s)

Huevos , Escherichia coli , beta-Lactamasas , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Animales , Huevos/microbiología , Turquía/epidemiología , Antibacterianos/farmacología , Pollos/microbiología , Farmacorresistencia Bacteriana/genética

RESUMEN

Serine ß-lactamases inactivate ß-lactam antibiotics in a two-step mechanism comprising acylation and deacylation. For the deacylation step, a water molecule is activated by a conserved glutamate residue to release the adduct from the enzyme. The third-generation cephalosporin ceftazidime is a poor substrate for the class A ß-lactamase BlaC from Mycobacterium tuberculosis but it can be hydrolyzed faster when the active site pocket is enlarged, as was reported for mutant BlaC P167S. The conformational change in the Ω-loop of the P167S mutant displaces the conserved glutamate (Glu166), suggesting it is not required for deacylation of the ceftazidime adduct. Here, we report the characterization of wild type BlaC and BlaC E166A at various pH values. The presence of Glu166 strongly enhances activity against nitrocefin but not ceftazidime, indicating it is indeed not required for deacylation of the adduct of the latter substrate. At high pH wild type BlaC was found to exist in two states, one of which converts ceftazidime much faster, resembling the open state previously reported for the BlaC mutant P167S. The pH-dependent switch between the closed and open states is caused by the loss at high pH of a low-barrier hydrogen bond, a proton shared between Asp172 and Asp179. These results illustrate how readily shifts in substrate specificity can occur as a consequence of subtle changes in protein structure.

Asunto(s)

Ácido Aspártico , Protones , beta-Lactamasas , Especificidad por Sustrato , beta-Lactamasas/química , beta-Lactamasas/metabolismo , beta-Lactamasas/genética , Concentración de Iones de Hidrógeno , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Conformación Proteica , Dominio Catalítico , Ceftazidima/química , Ceftazidima/metabolismo , Ceftazidima/farmacología , Cinética , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Mutación